**Marcelo S. da Silva \* , Marcela O. Vitarelli, Bruno F. Souza and Maria Carolina Elias \***

Laboratório de Ciclo Celular, Center of Toxins, Immune Response and Cell Signaling (CeTICS), Instituto Butantan, São Paulo 05503-900, Brazil; marcela.vitarelli@butantan.gov.br (M.O.V.); fsouza.bruno@gmail.com (B.F.S.)

**\*** Correspondence: mamasantos2003@yahoo.com.br (M.S.d.S.); carolina.eliassabbaga@butantan.gov.br (M.C.E.)

Received: 27 March 2020; Accepted: 5 May 2020; Published: 8 May 2020

**Abstract:** Single-celled eukaryote genomes predominantly replicate through multiple origins. Although origin usage during the S-phase has been elucidated in some of these organisms, few studies have comparatively approached this dynamic. Here, we developed a user-friendly website able to calculate the length of the cell cycle phases for any organism. Next, using a formula developed by our group, we showed a comparative analysis among the minimum number of replication origins (MO) required to duplicate an entire chromosome within the S-phase duration in trypanosomatids (*Trypanosoma cruzi*, *Leishmania major*, and *Trypanosoma brucei*) and yeasts (*Saccharomyces cerevisiae* and *Schizosaccharomyces pombe*). Using the data obtained by our analysis, it was possible to predict the MO required in a situation of replication stress. Also, our findings allow establishing a threshold for the number of origins, which serves as a parameter for genome approaches that map origins. Moreover, our data suggest that when compared to yeasts, trypanosomatids use much more origins than the minimum needed. This is the first time a comparative analysis of the minimum number of origins has been successfully applied. These data may provide new insight into the understanding of the replication mechanism and a new methodological framework for studying single-celled eukaryote genomes.

**Keywords:** trypanosomatids; yeasts; trypanosomatids genome; cell cycle phases; S-phase duration; DNA replication; replication origins
